Uromodulin processing in DNAJB11-kidney disease
Authors: Marta Mariniello, Guglielmo Schiano, Ayumi Yoshifuji, Valentine Gillion, John Andrew Sayer, François Jouret, Yannick Le Meur, Emilie Cornec-Le Gall, Eric Gregory Olinger, Olivier Devuyst
Journal: Kidney International (2023)

Autosomal dominant polycystic kidney disease (ADPKD) and autosomal dominant tubulointerstitial kidney disease (ADTKD) are among the most frequent monogenic disorders causing chronic kidney disease (CKD). The main genes associated with ADPKD are PKD1 and PKD2 encoding membrane and primary cilia proteins, polycystin-1 and polycystin-2, which are involved in multiple pathways regulating tubular cell differentiation. Recently, genes coding for proteins involved in the endoplasmic reticulum (ER) have been associated with rare, atypical forms of ADPKD. These genes include ALG5, ALG8 and ALG9, coding for enzymes involved in the lipid-linked oligosaccharide assembly for N-glycosylation of proteins; GANAB, coding for the α-subunit of the glucosidase II which removes a glucose residue from immature glycoproteins before they enter the calnexin/calreticulin folding and quality control cycle; and DNAJB11, which encodes a co-factor of BiP (binding immunoglobulin protein), a chaperone required for the proper folding and assembly of secreted and membrane proteins (Figure 1a). The characterization of 77 affected individuals from 27 families carrying heterozygous DNAJB11 variants revealed late-onset kidney failure, non-enlarged kidneys harbouring small cysts and interstitial fibrosis, and gout, suggesting partial overlap between DNAJB11-associated ADPKD and ADTKD.1Cornec-Le Gall E. Olson R.J. Besse W. et al.Monoallelic Mutations to DNAJB11 Cause Atypical Autosomal-Dominant Polycystic Kidney Disease.Am J Hum Genet. 2018; 102: 832-844https://doi.org/10.1016/j.ajhg.2018.03.013Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar,2Huynh V.T. Audrézet M.P. Sayer J.A. et al.Clinical spectrum, prognosis and estimated prevalence of DNAJB11-kidney disease.Kidney Int. 2020; 98: 476-487https://doi.org/10.1016/j.kint.2020.02.022Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar ADTKD is an increasingly recognized cause of kidney failure, characterized by tubular damage and interstitial fibrosis of the kidney in the absence of glomerular lesions. The most common gene associated with ADTKD is UMOD, coding for uromodulin - the most abundant protein excreted in normal urine. Uromodulin is a kidney-specific glycosylphosphatidylinositol (GPI)-anchored glycoprotein that is essentially produced by the epithelial cells lining the thick ascending limb (TAL) of the loop of Henle. The protein contains 616 amino acids, including 48 cysteine residues engaged in 24 intramolecular disulfide bonds, as well as 8 N-glycosylation sites. After proper maturation and apical targeting in TAL cells, uromodulin is cleaved by a serine protease and assembled in the urine into polymers that form a gel-like structure. The vast majority of disease-causing variants in UMOD causing ADTKD are heterozygous missense changes, often involving cysteine, that lead to the accumulation of uromodulin in the ER, damaging the TAL, inducing inflammation, and driving interstitial fibrosis (Figure 1a). ADTKD-UMOD is thus as a storage disease with a gain-of-toxic function, reflected by a sharp reduction of uromodulin excretion in the urine.3Devuyst O. Olinger E. Weber S. et al.Autosomal dominant tubulointerstitial kidney disease.Nat Rev Dis Primer. 2019; 5: 60https://doi.org/10.1038/s41572-019-0109-9Crossref PubMed Scopus (76) Google Scholar Recently, intermediate-effect variants in UMOD have been associated with subtle processing and maturation defects in vitro and intermediate urinary levels of uromodulin and milder kidney disease progression.4Olinger E. Schaeffer C. Kidd K. et al.An intermediate-effect size variant in UMOD confers risk for chronic kidney disease.Proc Natl Acad Sci U S A. 2022; 119e2114734119https://doi.org/10.1073/pnas.2114734119Crossref Scopus (12) Google Scholar The fact that DNAJB11 and its partner BiP assist in the folding of client proteins in the ER,5Shen Y. Hendershot L.M. ERdj3, a stress-inducible endoplasmic reticulum DnaJ Homologue, serves as a coFactor for BiP's Interactions with unfolded substrates.Mol Biol Cell. 2005 Jan; 16: 40-50https://doi.org/10.1091/mbc.e04-05-0434Crossref PubMed Scopus (0) Google Scholar led to suggest that DNAJB11-related disease could include features of ADTKD-UMOD caused by defective processing of uromodulin. This hypothesis was supported by possible intracellular accumulation of uromodulin in kidney biopsies from two DNAJB11-affected subjects.1Cornec-Le Gall E. Olson R.J. Besse W. et al.Monoallelic Mutations to DNAJB11 Cause Atypical Autosomal-Dominant Polycystic Kidney Disease.Am J Hum Genet. 2018; 102: 832-844https://doi.org/10.1016/j.ajhg.2018.03.013Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar However, such staining is operator-dependent, requiring rigorous positive and negative controls. Furthermore, accumulation of uromodulin might depend on the underlying UMOD mutation, and the availability of kidney biopsies in ADTKD-UMOD is restricted. In that context, the urinary levels of uromodulin, normalized against residual eGFR values, are considered as a robust marker of its processing in TAL cells.4Olinger E. Schaeffer C. Kidd K. et al.An intermediate-effect size variant in UMOD confers risk for chronic kidney disease.Proc Natl Acad Sci U S A. 2022; 119e2114734119https://doi.org/10.1073/pnas.2114734119Crossref Scopus (12) Google Scholar,6Olinger E. Hofmann P. Kidd K. et al.Clinical and genetic spectra of autosomal dominant tubulointerstitial kidney disease due to mutations in UMOD and MUC1.Kidney Int. 2020; 98: 717-731https://doi.org/10.1016/j.kint.2020.04.038Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar Previous studies have indeed demonstrated that modifications of the maturation and processing of uromodulin in the TAL cells are reflected by decreased urinary levels, which are proportional to the degree of protein maturation defect.4Olinger E. Schaeffer C. Kidd K. et al.An intermediate-effect size variant in UMOD confers risk for chronic kidney disease.Proc Natl Acad Sci U S A. 2022; 119e2114734119https://doi.org/10.1073/pnas.2114734119Crossref Scopus (12) Google Scholar,6Olinger E. Hofmann P. Kidd K. et al.Clinical and genetic spectra of autosomal dominant tubulointerstitial kidney disease due to mutations in UMOD and MUC1.Kidney Int. 2020; 98: 717-731https://doi.org/10.1016/j.kint.2020.04.038Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar Here, we measured the urinary levels of uromodulin in a cohort of patients harbouring DNAJB11 variants compared to healthy subjects and to patients with ADTKD-UMOD or ADPKD and used well-established assays to assess the maturation and excretion of uromodulin in DNAJB11-deficient kidney cells. Access to publicly available single nucleus RNA-sequencing data of the human kidney showed that DNAJB11 and UMOD are both highly expressed in the TAL cells, providing basis for potential interactions (Supplementary Figure S1a, S1b). We measured the levels of uromodulin in urine samples obtained from 10 individuals heterozygous for disease-causing variants in DNAJB11 (Supplementary Figure S2), using a well-established enzyme-linked immunosorbent assay (ELISA), compared to levels in patients with ADTKD-UMOD (n=65) or ADPKD (n=178), and a reference population (n=884) (Table 1). The range of urinary levels of uromodulin in subjects with DNAJB11 mutations was similar to those in the reference population and the ADPKD cohort, whereas dramatically lower values were detected in ADTKD-UMOD patients (Figure 1b). Immunoblotting analyses confirmed the unchanged levels of uromodulin in urine, with no evidence for any change in glycosylation patterns, suggesting that only the mature protein is excreted in the urine of DNAJB11 patients (Figure 1c, 1d).Table 1Characteristics of patient cohorts and reference population.NGender (M/F)Mean age (years)uUMOD (mg/g creat)eGFR (mL/min/1.73 m2)uUMOD/eGFR (Arbitrary units)DNAJB11105/550 ± 2033.5 ± 23.272 ± 370.45 ± 0.16ADTKD-UMOD6526/3938 ± 133.59 ± 3.6346 ± 240.07 ± 0.05ADPKD17895/8354 ± 1114.2 ± 9.744 ± 130.33 ± 0.2Normal subjects884424/46047 ± 1734.1 ± 20.797 ± 180.35 ± 0.2M, male; F, female; uUMOD, urinary uromodulin; eGFR, estimated glomerular filtration rate (CKD-EPI). Data expressed as mean ± SD. Open table in a new tab M, male; F, female; uUMOD, urinary uromodulin; eGFR, estimated glomerular filtration rate (CKD-EPI). Data expressed as mean ± SD. We next tested whether Dnjab11 downregulation affects uromodulin processing in kidney tubular cells. To this end, we used mouse inner medullary collecting duct cells (mIMCD) stably expressing GFP-tagged wild-type human uromodulin. We knocked down Dnajb11 by treating the cells with an adenovirus expressing a short hairpin RNA against mouse Dnajb11 (Ad-shDnajb11) whereas a scramble adenovirus (Ad-Scrmbl) was used as control (Supplementary Figure S3a). No differences in transduction efficiency were observed after 4 days silencing in Ad-Scrmbl and Ad-shDnajb11 treated cells and no toxicity effect was detected compared to untreated cells (Supplementary Figure S3b). Compared to Ad-Scrmbl treated cells, shRNA treatment led to a sharp decrease of Dnajb11 expression whereas no significant changes were detected in the expression of UMOD and other chaperone genes coding for DNAJB11 interaction partners (Hspa5, Dnajb4, Hsp90aa1 and Cryab) (Figure 2a; Supplementary Figure S3c). Immunoblot analysis showed that Dnajb11 silencing did not have any effect on uromodulin or on BiP levels, whereas it caused a sizeable upregulation of the water channel AQP2 (Figure 2b). Furthermore, Dnajb11 silencing was not reflected by any difference in the glycosylation pattern of uromodulin nor in its secretion in the apical medium (Figure 2b). These observations were confirmed by immunofluorescence analysis showing that trafficking of uromodulin to the plasma membrane was unaffected by Dnajb11 downregulation in UMOD-GFP cells (Figure 2c). The UMOD-GFP cells represent a viable system to study uromodulin trafficking, as it can be modulated using physical and chemical agents (Figure 2d). Indeed, following either incubation at 23 °C or treatment with nocodazole, a well-established microtubule-disrupting agent,7Vasquez R.J. Howell B. Yvon A.M. et al.Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro.Mol Biol Cell. 1997 Jun; 8: 973-985https://doi.org/10.1091/mbc.8.6.973Crossref PubMed Scopus (358) Google Scholar we observed a shift in uromodulin localization, from a predominantly plasma membrane-enriched signal to a more diffuse cytosolic vesicular pattern (Figure 2e, 2f). These results are in line with previous work showing that genetic disruption of the cytoskeletal network negatively impacts on uromodulin trafficking.8Joseph C.B. Mariniello M. Yoshifuji A. et al.Meta-GWAS Reveals Novel Genetic Variants Associated with Urinary Excretion of Uromodulin.J Am Soc Nephrol JASN. 2022; 33: 511-529https://doi.org/10.1681/ASN.2021040491Crossref PubMed Scopus (9) Google Scholar The fact that the excretion and maturation of uromodulin are unchanged in patients with DNAJB11 mutations contrasts with the strong decrease observed in patients with ADTKD-UMOD. Furthermore, the genetic downregulation of Dnajb11, to a level modelling haploinsufficiency, has no detectable impact on uromodulin maturation, trafficking and secretion in kidney tubular (mIMCD) cells. Since these cells are stably overexpressing uromodulin, one could expect that any effect of DNAJB11 silencing on uromodulin processing would be particularly noticeable. These findings are in line with recent studies showing no change in uromodulin processing in Dnajb11 knock-out mice. Instead, the loss of Dnajb11 was reflected by impaired cleavage of polycystin-1 and polycystin-1 dosage-dependent cystogenesis.9Ghosh R.S. Zhigui L. Guo Z. et al.Dnajb11-Kidney Disease Develops from Reduced Polycystin-1 Dosage but not Unfolded Protein Response in Mice.JASN. 2023; 34: 1521-1534https://doi.org/10.1681/ASN.0000000000000164Crossref Scopus (0) Google Scholar Together, these data do not support that defective maturation or processing of uromodulin is involved in ADPKD due to heterozygous DNAJB11 mutations. Yet, considering the role of DNAJB11 and BIP in the UPR,1Cornec-Le Gall E. Olson R.J. Besse W. et al.Monoallelic Mutations to DNAJB11 Cause Atypical Autosomal-Dominant Polycystic Kidney Disease.Am J Hum Genet. 2018; 102: 832-844https://doi.org/10.1016/j.ajhg.2018.03.013Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar,5Shen Y. Hendershot L.M. ERdj3, a stress-inducible endoplasmic reticulum DnaJ Homologue, serves as a coFactor for BiP's Interactions with unfolded substrates.Mol Biol Cell. 2005 Jan; 16: 40-50https://doi.org/10.1091/mbc.e04-05-0434Crossref PubMed Scopus (0) Google Scholar it is not excluded that variants in DNAJB11 could affect the progression in ADTKD-UMOD. In such scenario, loss-of-function DNAJB11 mutations would negatively impact on the function of BIP, potentially worsening ER stress due to uromodulin aggregates and accelerating organ damage in ADTKD-UMOD. That Dnajb11 silencing led to the upregulation of endogenous water channel AQP2 in mIMCD cells should also be noted, considering the role of the vasopressin V2R-cAMP and AQP2 pathway in ADPKD cystogenesis.10Devuyst O. Torres V.E. Osmoregulation, vasopressin, and cAMP signaling in autosomal dominant polycystic kidney disease.Curr Opin Nephrol Hypertens. 2013; 22: 459-470https://doi.org/10.1097/MNH.0b013e3283621510Crossref PubMed Scopus (59) Google Scholar The authors have declared no competing interest. M.M and O.D. are supported by the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant (agreement N° 860977). O.D. is supported by the European Reference Network for Rare Kidney Diseases (ERKNET, project N° 739532), the Swiss National Science Foundation (grant 310030-189044), and the University Research Priority Program (URPP) ITINERARE at the University of Zurich. A. Y. is supported by JSPS KAKENHI Grant Number JP22K16378. E.O. is supported by Postdoc Mobility-Stipend of the Swiss National Science Foundation Grants (P2ZHP3_195181 and P500PB_206851), Kidney Research UK Grant Paed_RP_001_20180925, and the Fonds National de la Recherche Luxembourg Grant 6903109. J.A.S. is supported by Kidney Research UK (Paed_RP_001_20180925 and RP_007_20210729) and the Northern Counties Kidney Research Fund (2019/01). The Genkyst study is supported by a National Plan for Clinical Research (PHRC inter-regional GeneQuest, NCT02112136, ECLG). We thank Aleksandra Kokanovic and Nadine Nägele for expert technical assistance; Inès Dufour for clinical information on ADTKD families; the German CKD study (Kai-Uwe Eckardt, Anna Köttgen, Heike Meiselbach) for the reference ADPKD population; and all participating patients and families. DATA SHARING STATEMENT All data are included in the article and/or supporting materials, including data [single nucleus RNA-seq for UMOD and DNAJB11 in human kidney; DNAJB11 mutations annotation; and interaction network of DNAJB11] derived from resources available in the public domain: [Cellxgene repository (https://cellxgene.cziscience.com/collections/b3e2c6e3-9b05-4da9-8f42-da38a664b45b); Ensembl canonical transcript for DNAJB11 (ENST00000265028.8); https://string-db.org)]. Additional information can be provided on request to the principal investigators of the study. Download .docx (.75 MB) Help with docx files